Unlike B and T cells, NK cells do not express antigen-specific receptors, yet they can eliminate virus-infected cells and cancer cells without harming normal cells. An important component in the specific recognition of target cells by NK cells are NK cell inhibitory receptors that recognize surface molecules called major histocompatibility complex (MHC) class I. MHC-specific recognition by inhibitory receptors on NK cells prevents the killing of normal, healthy cells. The major goal of this project is to elucidate the mechanism by which inhibitory receptors block NK cell activation. Specific recognition of target cells is controlled by combinations of germ-line encoded receptors. How activation is achieved and controlled during encounter of NK cells with target cells is still unclear. Cytotoxicity of NK cells towards target cells is governed by signals transmitted through multiple receptor-ligand interactions. Activation of freshly isolated, primary NK cells is tightly regulated and requires signaling by the combination of synergistic receptors, which are not activating on their own. The main questions addressed here concerned the basis for the requirement of co-activation receptor synergy, and the sensitivity of granule polarization and degranulation to inhibitory receptors. Is the requirement for synergy due to the control by distinct receptors of independent steps in the activation pathway, or to a proximal convergence of signals, which may serve to override an early activation threshold? Using co-engagement of receptors NKG2D and 2B4 as a model, we have found that they synergize at a very early activation step, at the level of, or upstream of PLC-gamma activation, which is completely dependent on Vav1. Furthermore, synergy is required to overcome an activation threshold set by c-Cbl. These data were obtained using either primary, resting NK cells or an NK cell line that had been rested for a day before use. Stimulation was triggered by crosslinking of receptors with Abs, and was also evaluated in the more physiological context of receptorligand interaction between NK cells and target cells. The results have revealed an unsuspected similarity between innate NK cell responses and the priming of nave T cells. For the latter, full activation requires costimulation by CD28 in order to overcome inhibition by Cbl-b, whereas NK cells require a synergy of co-activation receptors to overcome inhibition by c-Cbl. c-Cbl inhibited Vav1-dependent signals, given that c-Cbl knockdown did not rescue the Vav1 defect. Moreover, c-Cbl knockdown and Vav1 overexpression each circumvented the necessity for synergy because NKG2D or 2B4 alone became sufficient for activation. Thus, synergy requires not strict complementation but, rather, strong Vav1 signals to overcome inhibition by c-Cbl. Inhibition of NK cell cytotoxicity by CD94-NKG2A binding to HLA-E on target cells was dominant over synergistic activation, even after c-Cbl knockdown. Therefore, NK cell cytotoxicity is tightly controlled at the level of Vav1 by a hierarchy of inhibitory mechanisms. Inhibition of NK cell responses by ITIM-containing receptors occurs at a proximal step, upstream of Ca2+ mobilization and of actin polymerization-dependent processes. The identification of Vav1 as a primary substrate for dephosphorylation by SHP-1 during inhibitory receptor engagement by MHC class I on target cells, and as an essential component in the NKG2D and 2B4 synergy provides an explanation for the dominance of inhibitory receptors over synergistic activation signals. Our results have revealed a hierarchy of inhibitory signals to control NK cell activation: a Vav1-dependent synergy is required to overcome inhibition by c-Cbl but is dominantly inhibited by CD94-NKG2A, even when inhibition by c-Cbl had been lifted. Natural cytotoxicity is achieved by polarized release of perforin and granzymes at the NKtarget cell immunological synapse. Signals for granule polarization and degranulation can be uncoupled in NK cells, which raises the question of their respective sensitivity to inhibitory receptors. Expression of either HLA-C or HLA-E on the human cell line 721.221 blocked granule polarization, degranulation, and CD16-dependent MIP-1alpha secretion by NK cell clones that expressed inhibitory receptors of matching HLA specificity. To test inhibition of signals for polarization and degranulation separately, Drosophila S2 cells expressing ICAM-1 with either HLA-C or HLA-E were used. CD16-dependent degranulation and MIP-1alpha secretion were not fully inhibited, suggesting that other receptorligand interactions, which occur with 721.221 cells, contribute to inhibition. In contrast, HLA-C or HLA-E on S2 cells co-expressing ICAM-1 or ULBP1 were sufficient to block granule polarization induced by either LFA-1 or NKG2D, even during concomitant CD16-dependent degranulation. Similarly, expression of a ligand for NKR-P1A on S2 cells inhibited granule polarization, but not CD16-induced degranulation. Therefore, granule polarization, rather than degranulation, is the preferred target of inhibitory receptors in NK cells. The main conclusion is that inhibitory receptors are better equipped to stop granule polarization than to block GrzB and chemokine release. This was shown definitively by observing sustained GrzB and MIP-1&#945;release by NK cells in which granule polarization was inhibited. The evidence obtained for inhibition of NK cells by HLA class I on Drosophila S2 cells is conclusive, because inhibition is dependent on addition of HLA class I-specific peptides. Persistent degranulation during inhibition of polarization in IL-2-activated NK cells suggests that it may occur in high inflammatory conditions. Prevention of NK cell cytotoxicity would be better achieved through inhibition of degranulation rather than polarization. However, the possibility of releasing the block in degranulation while maintaining inhibition of polarization endows NK cells with the potential to provide bystander killing while still refraining from direct attacks on MHC class I-positive cells.